Platelets arise from the fragmentation of megakaryocytes, which are large polyploid bone marrow cells produced by several cycles of chromosomal duplication without cytoplasmic division (Handin (Wilson et. al., eds) in Harrison's Principles of Internal Medicine 12th edition (1991)). Once free of the marrow space, approximately 2/3 of the platelets circulate freely, while approximately 1/3 are sequestered in the spleen. Circulating platelets last for 7 to 10 days, after which they are removed by phagocytic cells. A decrease in platelet mass stimulates megakaryocytopoiesis, resulting in an increase in the number, size and ploidy of the megakaryocytes.
Platelet receptors which mediate platelet adhesion and aggregation are located on the two major platelet surface glycoprotein complexes. These complexes are the glycoprotein Ib-IX complex which facilitates platelet adhesion by binding von Willebrand factor (vWF), and the glycoprotein IIb-IIIa complex which links platelets into aggregates by binding to fibrinogen. Patients with the Bernard Soulier syndrome, a congenital bleeding disorder, show deficient platelet adhesion due to a deficiency in the glycoprotein Ib-IX complex which binds vWF, mild thrombocytopenia, and large lymphocoid platelets.
Glycoprotein v (GPV) is a major (.apprxeq.12,000 molecules/platelet), heavily glycosylated platelet membrane protein (Mr 82,000) (Modderman et. al. J. Biol. Chem. 267: 364-369). Earlier reports showing that GPV was a peripheral protein (Berndt and Phillips J. Biol. Chem 256: 59-65) were presumably due to the release of GPV from the membrane by calpain during the purification procedure. Exposure of platelets to thrombin liberates a 69 kDa soluble fragment termed GPVf1 (Phillips and Poh-Agin, Biochem. Biophys. Res. Commun. 75: 940-947). This, and its absence in the Bernard-Soulier syndrome (Clemetson) et. al., J. Clin. Invest. 70: 304-311 (1982); Nurden et. al., J. Clin. Invest. 67: 1431 (1981); Berndt et. al., Blood 62: 800-807 (1983)), led to the suggestion that GPV may be involved in the thrombin-induced activation response (Berndt and Phillips J. Biol. Chem 256: 59-65 (1981)). Recent experiments show that GPV can interact non-covalently with the GPIb-IX complex (Modderman et. al. J. Biol. Chem. 267: 364-369) 1992, a complex formed by the non-covalent association of GPIb (consisting of GPIb.alpha., a 145 kDa protein, disulfide linked to GPIb.beta., a 24 kDa protein) with GPIX (a 22 kDa protein). The binding sites for von Willebrand factor and for thrombin on the GPIb-IX complex have been localized on GPIb.alpha. (Wicki and Clemetson Eur. J. Biochem. 153: 1-11 (1985); Vicente et. al., J. Biol. Chem. 265: 274-280 (1990)). Since thrombin is now known to activate platelets by cleaving the thrombin receptor (Vu et. al., Cell 64 1057-1068 (1991)), a G-protein coupled receptor, it is unknown whether thrombin cleaves GPV incidently as a consequence of thrombin binding to GPIb.alpha., or whether this cleavage has a physiological role.
The amino acid sequences of GPIb.alpha., GPIb.beta., and GPIX have been deduced from their cDNA and genomic sequences (Lopez et. al., Proc. Natl. Acad. Sci. USA 84: 5614-5619 (1987); Wenger et. al., Biochem. Biophys. Res. Commun. 156: 389-395 (1988); Lopez et. al., Proc. Natl. Acad. Sci. USA 85: 2135-2139 (1988); Hickey, et. al., Proc. Natl. Acad. Sci. USA 86: 6733-6777 (1989); Hickey and Roth J. Biol. Chem 268: 3438-3443 (1993)). Analysis of the primary amino acid sequence of GPIb.alpha., GPIb.beta., and GPIX has revealed a common evolutionary origin for the three proteins, as they contain one or more homologous 24 amino acid leucine-rich domain. These domains are also found in a large family of leucine-rich glycoproteins (LRG) including leucine-rich .alpha.2 GP, proteoglycan core, fibromodulin, human lutropin-chorio gonatropin receptor and RNAse inhibitor, and toll protein and chaoptin found in Drosophila (reviewed in Roth Blood 77: 5-19 (1991)). Recently, analysis of partial peptide sequences obtained from purified platelet GPV suggested that GPV is also a member of the LRG family (Shimomura et. al., Blood 75: 2349-2356 (1990); Roth et. al., Biochem Biophys. Res. Commun 170: 153-161 (1990)).
GPV is a very specific marker for the megakaryocytic cell lineage. A monoclonal antibody specific for GPV (SW16) was recently shown to bind exclusively to platelets (Modderman et. al., J. Biol. Chem. 267: 364-369 (1992)). SW16 did not bind to red cells, leukocytes, endothelial cells, or cell lines such as HEL or MEG-01 which are known to express platelet megakaryocyte markers.